Human 'Mini-Brain'.....

             Cerebral Organoids produced in a laboratory!

    Science 30 August 2013: 
    Vol. 341 no. 6149 pp. 946-947 
    DOI: 10.1126/science.341.6149.946

No bigger than apple seeds, the cell clusters are simply referred to as "cerebral organoids." But that careful language in a paper in this week's issue of Nature belies the excitement of many neuroscientists at what it reports: the growth from human embryonic stem cells of semiorganized knots of neural tissue that contain the rudiments of key parts of the human brain, including the hippo campus and prefrontal cortex.

"I find it amazing," says Wieland Huttner, a neuroscientist at the Max Planck Institute of Molecular Cell Biology and Genetics in Dresden, Germany, who was not involved in the study. "It's not real brain—that's clear. But I'm positively surprised that so many features are reproduced." Developmental geneticist Madeline Lancaster and her colleagues, who grew the organoids in a Vienna laboratory, have already shown that they can use the organoids to probe how normal human brain development goes awry in a genetic brain disorder.

Left to their own devices in a lab dish, embryonic stem (ES) cells will differentiate into a menagerie of tissues: beating heart cells, neurons, even hair and teeth. The trick for scientists has been to harness that potential, coaxing the cells to grow into the kinds of tissues they want to study or use for a therapy.

Developmental biologists know that neural tissue is a sort of default fate for differentiating embryonic cells, and researchers have been able to grow a variety of specific neural cell types from ES cells. But the level of cellular organization seen in this latest brain-in-a-dish paper is a significant step forward, says Magdalena Götz, who studies neurodevelopment at the Ludwig Maximilian University of Munich in Germany.

Lancaster's work took place in the lab of Jürgen Knoblich, a developmental geneticist at the Institute of Molecular Biotechnology of the Austrian Academy of Science in Vienna. It exploits what Knoblich calls the "absolutely enormous self-organizing capacity of developing human cells. If you just leave them alone and provide a medium that is supportive enough, they do things on their own." It also builds on work by Yoshiki Sasai of the RIKEN Center for Developmental Biology in Kobe, Japan. In 2008, he and his colleagues reported that mouse and human ES cells in culture could spontaneously form cell layers that resemble the cortical layers in the brain.

Lancaster, a postdoctoral researcher in Knoblich's lab, was trying to culture early neural tissue to better understand how and when developing brain cells switch from proliferation—making more of themselves—to differentiation—making more mature cell types, which don't continue to divide. Lancaster started with techniques developed by Sasai and others that shepherd dividing stem cells toward a neural fate, but she was also intrigued, she says, by the "miniguts" that another research team had grown in droplets of Matrigel, a gelatinous protein mixture that can help cells grow in three dimensions. So she embedded clusters of stem cell–derived neural cells in a droplet of the material (see diagram).

The Matrigel droplets freed the cell clusters to grow larger and develop more complex structures, without any further coaxing. To increase the availability of oxygen and other nutrients to the inner layers of the structures, Lancaster put the Matrigel droplets into a slowly rotating bioreactor, which gently shakes them.

Within a few weeks, Lancaster says, she noticed darker pigmented patches on some of the cell clusters. On closer inspection, she recognized the rudiments of eye tissue, a sign that more complex structures might be forming. When she described the data at a lab meeting, Knoblich says, "I was completely blown away. I couldn't sleep that night."

When lab members looked inside some of the cerebral organoids, they found structures that resemble the choroid plexus (the cavity in the brain that produces cerebrospinal fluid), the cerebral cortex (the brain's outermost layer), and retinal tissue. More detailed staining showed evidence that after 16 days of development, the organoids had what resembles forebrain, midbrain, and hindbrain regions. The team also found molecular markers for a variety of more specialized regions—including the outer subventricular zone (OSVZ), a feature of human, but not mouse, brains. Organoids grown from mouse ES cells did not develop an OSVZ region.

The resemblance to a real brain only goes so far. The organoids do not have any blood vessels, so cells at their core die. They reach their maximum size—about 3 millimeters in diameter—after 2 to 3 months, Lancaster says, and after 4 months they don't develop any new cell types. However, the cell clusters can apparently survive indefinitely in the bioreactor; the oldest ones have been in culture for nearly a year, the researchers report.

The cerebral organoids may shed light on human brain diseases that are difficult to study in mice or other animals. For example, the scientists used the structures to study microcephaly, a neurodevelopmental disorder in which the head and brain end up much smaller than normal. Rather than starting with ES cells, they took cells from a person with a particular form of the disorder and "reprogrammed" them into so-called induced pluripotent stem (iPS) cells. Mice are a poor model for that form of microcephaly; the specific genetic mutation responsible results in mice with brains that are only slightly smaller than normal. In contrast, organoids derived from the patient's iPS cells were shrunken, and Knoblich's team found a clue to why. Certain precursor cells were maturing earlier than normal, bringing tissue growth to a halt prematurely.

The organoids are probably not yet useful for studying more complex neurodevelopmental conditions such as autism or schizophrenia, because those conditions involve more mature cells and complex cell connections. Lancaster and Knoblich also note that each organoid develops distinctively, resulting in significant differences in composition and structure that make it hard to do controlled experiments.

The researchers are working on ways to grow more consistent organoids—and to incorporate some sort of vascular system so that the cell clusters can grow bigger and presumably develop further. They hope that additional teams will take up and improve the method. Götz and others says they plan to do just that. "For studying the cerebral cortex, this is the best model so far," she says. "People will use it, and time will tell how useful it is."